Glycosidases of Ehrlich Ascites Tumor Cells and Ascitic Fluid-Purification and Substrate Specificity of &V-Acetylgalactosaminidase and a-Galactosidase: Comparison with Coffee Bean a-Galactosidase’

نویسندگان

  • Fumio Yagi
  • Allen E. Eckhardt
  • Irwin J. Goldstein
چکیده

Ehrlich ascites tumor cells and ascitic fluid were assayed for glycosidase activity. cu-Galactosidase and P-galactosidase, (Yand @-mannosidase, a-N-acetylgalactosaminidase, and /3-N-acetylglucosaminidase activities were detected using p-nitrophenyl glycosides as substrates. a-Galactosidase and a-N-acetylgalactosaminidase were isolated from Ehrlich ascites tumor cells on t-aminocaproylgalactosylamine-Sepharose. (YGalactosidase was purified 160,000-fold and was free of other glycosidase activities. cy-N-Acetylgalactosaminidase was also purified 160,000-fold but exhibited a weak a-galactosidase activity which appears to be inherent in this enzyme. Substrate specificity of the a-galactosidase was investigated with 12 substrates and compared with that of the corresponding coffee bean enzyme. The pH optimum of the Ehrlich cell cY-galactosidase centered near 4.5, irrespective of substrate, whereas the pH optimum of the coffee bean enzyme for PNP-a-Gal was 6.0, which is 1.5 pH units higher than that for other substrates of the coffee bean enzyme. The reverse was found for a-N-acetylgalactosaminidase: the pH optimum for the hydrolysis of PNP-a-GalNAc was 3.6, lower than the pH 4.5 required for the hydrolysis of GalNAccu1,3Gal. Coffee bean cY-galactosidase showed a relatively broad substrate specificity, suggesting that it is suited for cleaving many kinds of terminal a-galactosyl linkages. On the other hand, the substrate specificity of Ehrlich a-galactosidase appears to be quite narrow. This enzyme was highly active toward the terminal a-galactosyl linkages of Ehrlich glycoproteins and laminin, both of which possess Galal,

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تاریخ انتشار 2003